Levels Of Luciferase Activity Research Articles

Abstract 15042: Cardiac-Enhanced Adeno-Associated Viral Vector Achieves Long-Term Transgene Expression in Transplanted Porcine Hearts When Administered During Ex Vivo Perfusion

Introduction: Use of gene therapy for cardiac transplantation is limited by the lack of an effective recombinant Adeno-associated viral vector (rAAV) gene delivery strategy. Ex vivo normothermic perfusion has been increasingly used in clinical practice and is an effective platform for achieving robust global gene delivery to a donor heart. Here we utilized a rationally designed cardiac-enhanced AAV3b variant, SASTG, to deliver the gene firefly luciferase to porcine hearts and evaluated transgene expression 30-days post-transplantation. Methods: Heterotopic heart transplantation was performed using blood-type and MHC matched Yucatan pigs (n=4). Donor hearts underwent ex vivo perfusion for 2-hours with perfusate containing the rAAV SASTG-Luciferase. Three different doses of rAAV were used: 2x10 13 , 8x10 13 , 1x10 14 viral genome copies (VGC). Recipients were survived for 30 days at which time they were euthanized and donor and recipient hearts were examined for overall levels of luciferase activity, luciferase DNA copy number, and histology for luciferase expression on immunostaining and evidence of inflammation. Results: There was a dose-dependent response in luciferase activity observed in the donor heart ventricles with the lowest activity detected at the 2x10 13 VGC dose (1.5-9.9x10 3 RLU/mg) mid-level activity at the 8x10 13 VGC dose (32.7-71.2x10 3 RLU/mg) and highest activity at the 1x10 14 VGC dose (27.6-278.1x10 3 RLU/mg). Luciferase DNA was detectable in a dose-dependent manner in the donor heart ventricles (0.02-131.1x10 3 VGC/pg of DNA), but undetectable in all native hearts. Finally, immunostaining demonstrated diffuse luciferase expression in the donor heart but none in the recipient hearts. There was also no evidence of inflammation in any of the donor hearts. Conclusions: The cardiac-enhanced AAV3b variant, SASTG, is associated with robust, global transgene expression in porcine cardiac allografts when administered during ex vivo perfusion.

Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress

BackgroundA reasonable supply of nitrogen (N) fertilizer is essential for obtaining high-quality, high-level, and stable potato yields, and an improvement in the N utilization efficiency can effectively reduce N fertilizer use. It is important to use accurate, straightforward, and efficient transgenic breeding techniques for the identification of genes that can improve nitrogen use efficiency, thus enabling us to achieve the ultimate goal of breeding N-efficient potato varieties. In recent years, some of the mechanisms of miRNAs have been elucidated via the analysis of the correlation between the expression levels of potato miRNA target genes and regulated genes under conditions of stress, but the role of miRNAs in the inhibition/expression of key genes regulating N metabolism under N stress is still unclear. Our study aimed to identify the role played by specific enzymes and miRNAs in the responses of plants to N stress.ResultsThe roots and leaves of the N-efficient potato variety, Yanshu4 ("Y"), and N-inefficient potato variety, Atlantic ("D"), were collected at the seedling and budding stages after they were exposed to different N fertilizer treatments. The miRNAs expressed differentially under the two types of N stress and their corresponding target genes were first predicted using miRNA and degradome analysis. Then, quantitative polymerase chain reaction (qRT-PCR) was performed to verify the expression of differential miRNAs that were closely related to N metabolism. Finally, the shearing relationship between stu-miR396-5p and its target gene StNiR was determined by analyzing luciferase activity levels. The results showed that NiR activity increased significantly with an increase in the applied N levels from the seedling stage to the budding stage, and NiR responded significantly to different N treatments. miRNA sequencing enabled us to predict 48 families with conserved miRNAs that were mainly involved in N metabolism, carbon metabolism, and amino acid biosynthesis. The differences in the expression of the following miRNAs were identified via screening (high expression levels and P < 0.05): stu-miR396-5p, stu-miR408b-3p_R-1, stu-miR3627-3p, stu-miR482a-3p, stu-miR8036-3p, stu-miR482a-5p, stu-miR827-5p, stu-miR156a_L-1, stu-miR827-3p, stu-miR172b-5p, stu-miR6022-p3_7, stu-miR398a-5p, and stu-miR166c-5p_L-3. Degradome analysis showed that most miRNAs had many-to-many relationships with target genes. The main target genes involved in N metabolism were NiR, NiR1, NRT2.5, and NRT2.7. qRT-PCR analysis showed that there were significant differences in the expression levels of stu-miR396-5p, stu-miR8036-3p, and stu-miR482a-3p in the leaves and roots of the Yanshu4 and Atlantic varieties at the seedling and budding stages under conditions that involved no N and excessive N application; the expression of these miRNAs was induced in response to N stress. The correlation between the differential expression of stu-miR396-5p and its corresponding target gene NiR was further verified by determining the luciferase activity level and was found to be strongly negative.ConclusionThe activity of NiR was significantly positively correlated with N application from the seedling to the budding stage. Differential miRNAs and target genes showed a many-to-many relationship with each other. The expression of stu-miR396-5p, stu-miR482a-3p, and stu-miR8036-3p in the roots and leaves of the Yanshu4 and Atlantic varieties at the seedling and budding stages was notably different under two types of N stress. Under two types of N stress, stu-miR396-5p was down-regulated in Yanshu4 in the seedling-stage and shoot-stage roots, and up-regulated in seedling-stage roots and shoot-stage leaves; stu-miR482a-3p was up-regulated in the seedling and shoot stages. The expression of stu-miR8036-3p was up-regulated in the leaves and roots at the seedling and budding stages, and down-regulated in roots under both types of N stress. The gene expressing the key enzyme involved in N metabolism, StNiR, and the stu-miR396-5p luciferase assay reporter gene had a strong regulatory relationship with each other. This study provides candidate miRNAs related to nitrogen metabolism and highlights that differential miRNAs play a key role in nitrogen stress in potato, providing insights for future research on miRNAs and their target genes in nitrogen metabolic pathways and breeding nitrogen-efficient potatoes.

Identification of Genetic and Chemical Factors Affecting Type III Secretion System Expression in Pseudomonas syringae pv. actinidiae Biovar 3 Using a Luciferase Reporter Construct.

The type III secretion system (T3SS) is a key factor in the pathogenesis of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), the causal agent of a global kiwifruit bacterial canker pandemic. To monitor the T3SS expression levels in Psa3, we constructed a luciferase reporter plasmid-expressing HrpAPsa3-NLuc fusion protein. The expression of HrpA-NLuc was induced in hrp-inducing conditions whereas the level of luciferase activity correlated with the expression of hrp/hrc genes in Psa3 confirmed the reliability of the reporter construct. Based on the readout of the NLuc reporter construct, three small molecule compounds 4-methoxy-cinnamic acid, sulforaphane, and ferulic acid were determined as T3SS inhibitors in Psa3, whereas sodium acetate was determined to be a T3SS inducer. Moreover, the aqueous extract of fruit inhibited the accumulation of HrpA-NLuc in Psa3 in medium and in planta. Additionally, the T3SS inhibitors suppress Psa3 virulence, whereas the T3SS inducer promotes Psa3 virulence on kiwifruit. Thus, our findings may provide clues to why the fruit is not infected by Psa3, and the Psa3 T3SS inhibitors have potential as alternatives to current nonspecific antimicrobials for disease management.

Abstract 5527: A novel fully human anti-TIGIT and PVRIG bispecific antibody that elicits potent anti-tumor efficacy in pre-clinical studies

Abstract Background: TIGIT (T-cell immunoglobulin and ITIM domain), which is primarily expressed on activated and 'exhausted' T and NK cells, is one of the promising 'next generation' immune checkpoint molecules. Engagement of TIGIT to its ligands (i.e., PVR and PVRL2) leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Anti-TIGIT monoclonal antibodies have shown clinical benefit when combined with anti-PD-L1 agents in NSCLC. However, the single-agent efficacy of anti-TIGIT therapies have been limited. PVRIG (PVR-related immunoglobulin domain containing), which is another coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family, binds with high affinity to PVRL2 and suppresses T-cell function, and shows nonredundant inhibitory effects alongside the TIGIT/PVR/PVRL2 axis. Here, we report a fully-human anti-TIGIT × PVRIG bispecific antibody (anti-TIGIT × PVRIG biAb), which blocks both the PVRIG/PVRL2 and TIGIT/PVR/PVRL2 pathways, that maintains the efficacy of the combination of the two mono-agents. The anti-TIGIT × PVRIG biAb is also highly efficacious when combined with PD1/PD-L1 inhibitors in mouse tumor models. Methods: An anti-TIGIT × PVRIG biAb was generated through the fusing of a fully-human IgG targeting TIGIT with a wild type G1-Fc to a fully-human scFv at the c-terminus targeting PVRIG. Binding affinity and specificity analyses were studied by flow cytometry and biolayer interferometry. The co-binding of the anti-TIGIT × PVRIG biAb to TIGIT and PVRIG was detected by ELISA. The immunomodulatory functions of the anti-TIGIT × PVRIG biAb were evaluated using a luciferase reporter cell assay in vitro and human PBMC-based tumor models in vivo. Results: The anti-TIGIT × PVRIG biAb binds with high affinity to the extracellular domain of human TIGIT/PVRIG and can bind to TIGIT and PVRIG simultaneously. In a competition assay, the anti-TIGIT × PVRIG biAb efficiently blocked the interaction between TIGIT and PVR/PVRRL2, and PVRIG with PVRL2. In a luciferase reporter cell system, the anti-TIGIT × PVRIG biAb induced high levels of luciferase activity compared with the anti-TIGIT or anti-PVRIG mAbs alone. In vivo, the anti-TIGIT × PVRIG biAb demonstrated stronger anti-tumor efficacy than the anti-TIGIT and anti-PVRIG mAbs as monotherapies or combined with anti-PD-1 mAb. Conclusion: Our anti-TIGIT × PVRIG biAb, a fully human bispecific antibody, either alone or in combination with anti-PD-1 mAb promotes immune cell activation both in vitro and in vivo, supporting its clinical development for the treatment of human cancers. The molecule is currently under GLP-toxicity evaluation in NHP, and a first-in-human study is expected to begin in 2022. Citation Format: Shuang Dai, Weifeng Huang, Zhijun Yuan, Shaogang Peng, Jiayi Si, Chao Wang, Xiaoniu Miao, Yingda Xu, Joanne Sun, Xiaolin Liu, Andy Tsun, Tianhang Zhai. A novel fully human anti-TIGIT and PVRIG bispecific antibody that elicits potent anti-tumor efficacy in pre-clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5527.

Identification of Cryptic Promoter Activity in cDNA Sequences Corresponding to PRRSV 5' Untranslated Region and Transcription Regulatory Sequences.

To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5′ untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5′ UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5′ UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

Construction of a Recombinant Porcine Epidemic Diarrhea Virus Encoding Nanoluciferase for High-Throughput Screening of Natural Antiviral Products.

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

MiR-373 Inhibits the Epithelial-Mesenchymal Transition of Prostatic Cancer via Targeting Runt-Related Transcription Factor 2.

Prostatic cancer (PCa) is a prevalent form of malignancy based on its high associated levels of mortality and morbidity across the world. MicroRNAs (miRNAs) are significant in the advancement of prostatic cancer. The current study is aimed at exploring the potential roles of miR-373 in PCa. In turn, the study conducted a qRT-PCR test to determine the levels of mRNA. A western blot test was also executed in determining the protein level. The processes of transwell assay and wound healing were integrated in the detection of the potential for PCa cells to invade and migrate. The integration of dual luciferase reporter assay is critical in determining the levels of luciferase activity among prostatic cancer cells. Then, the results showed a net decrease of miR-373 within prostatic cancer cells and tissues. Upregulated miR-373 reduced the invasion and migration potential of PCa cells. Moreover, overexpressed miR-373 increased the levels of E-cadherin and FSP1 as epithelial cell markers. Similarly, the overregulation of miR-373 brought about the upregulation of mesenchymal markers (N-cadherin, Snail, and vimentin). The study predicted runt-related transcription factor 2 (RUNX2) to be a target of miR-373. The luciferase activity of PCa cells was decreased after the cotransfection with miR-373 mimics and RUNX2 3′ untranslated region (3′UTR) wild type (WT). Moreover, RUNX2 became upregulated in PCa cells and tissues. The upregulation of miR-373 decreased the mRNA and protein level of RUNX2. However, overexpressed RUNX2 abated the roles of miR-373 in the intrusion and migration of PCa cells and in regulating the expression of epithelial cell markers and mesenchymal markers. In short, miR-373 may regulate the EMT of PCa cells via targeting RUNX2. The miR-373/RUNX2 axis provides a therapeutic target for PCa.

PD-1H (VISTA) Induces Immune Evasion in Acute Myeloid Leukemia

Despite progress in understanding the molecular pathogenesis in acute myeloid leukemia (AML), the disease remains highly fatal with limited therapeutic options. There is a need for novel approaches for management of AML that can overcome resistant disease. Immune checkpoint inhibitor therapy has emerged as a successful treatment strategy in several solid tumors and refractory Hodgkin lymphoma. However, preliminary data from clinical trials to date indicate that anti-PD-1 antibodies have limited single-agent activity in AML. This suggests that co-inhibitory molecules other than PD-1 may induce immune evasion in AML. Programmed Death-1 Homolog (PD-1H, VISTA) is a novel co-inhibitory molecule that induces immune evasion in solid tumors. Interestingly, The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia database both reveal that PD-1H is over-expressed in AML at the transcript level. While we found that myeloid subsets and T cells have the expression of PD-1H in bone marrow of healthy donors, we demonstrated that PD-1H protein is highly expressed in bone marrow of 8 (of 10) human AML donors using immunohistochemistry. To determine whether the high expression of PD-1H in AML bone marrow is associated with the expression of PD-1H in AML blasts, flow cytometric analyses were performed in bone marrow aspirate of human CD34+ AML donors. Mean Fluorescent Intensity (MFI) of CD34+ AML blasts was significantly higher when compared to normal CD34+ progenitor cells (MFI: 257±82 (AML CD34+ blasts) vs 10±4 (normal CD34+ progenitor cells), N=6, p<0.05). To test our hypothesis that PD-1H induces immune evasion in AML, we transplanted PD-1H-expressing murine myeloid leukemia cells (C1498) into syngeneic PD-1H knockout (KO) or wild type (WT) littermate mice. Whole body bioluminescence (unit: radiance) was used to determine the level of luciferase activity reflecting in vivo growth of luciferase expressing C1498 cells and survival of recipient PD-1H KO mice was compared to WT controls. We observed that murine AML cell growth in vivo was diminished in PD-1H KO mice (mean radiance in one representative experiment: 2.6x106 (KO) vs 4.2x107 (WT) on day 26, N=5, p<0.05) and survival of PD-1H KO mice was improved compared to WT controls (median survival: 35 days (KO) vs 28 days (WT), N=5, p<0.05). In addition, PD-1H antibody (Ab) decreased AML cell growth in vivo in KO mice (mean radiance in one representative experiment: 2.4x105 (KO + PD-1H Ab) vs 2.6x106 (KO + Isotype control) on day 26, N=5, p<0.05) and extended survival in C1498-bearing PD-1H KO mice (median survival: 45 days (KO + PD-1H Ab) vs 35 days (KO + Isotype), N=5, p<0.05). These data suggest that PD-1H on both host cells and AML cells induces immune evasion in AML. Based on prior preclinical models, we further hypothesized that immunogenicity of AML can be enhanced by epigenetic modulation, that can potentiate anti-leukemic effect of PD-1H blockade. We found that DNA methyltransferase inhibition (DNMTi) by 5-aza-2'-deoxycytidine (decitabine) increased T cell infiltration in subcutaneously injected murine AML (C1498) tumor (CD3+ cells/tumor cells: 33±5% (DNMTi) vs 10±3% (PBS control), N=3, p<0.05) using flow cytometry and immunohistochemistry. Importantly, DNMTi potentiated the anti-leukemic effect of the PD-1H blockade (mean radiance in one representative experiment: 9.9x108 (PBS + WT) vs 1.2x108 (PBS + KO) vs 3.6x105 (decitabine + WT) vs 6.9x104 (decitabine + KO) on day 21; 5.9x109 (PBS + WT) vs 3.5x108 (PBS + KO) vs 1.8x106 (decitabine + WT) vs 1.3x105 (decitabine + KO) on day 24, N=7, p<0.05) and significantly prolonged survival in PD-1H KO mice (median survival: 28 days (WT) vs 35 days (KO) vs 39 days (DNMTi + WT) vs 60 days (DNMTi + KO), p<0.05). Together, our data suggest that PD-1H is highly expressed in human AML and can induce immune evasion in a murine model of AML (C1498). DNMTi has potential synergistic effects on PD-1H blockade and combining these two can be an important immunotherapeutic approach in AML. DisclosuresZeidan:Otsuka: Consultancy; AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Takeda: Speakers Bureau. Chen:Boehringer Ingelheim, Pfizer, NextCure: Research Funding.

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors.

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

Effects of Naturally Occurring Mutations in Bovine Leukemia Virus 5'-LTR and Tax Gene on Viral Transcriptional Activity.

Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis (EBL). The long terminal repeat (LTR) plays an indispensable role in viral gene expression. The BLV Tax protein acts as the main transactivator of LTR-driven transcription of BLV viral genes. The aim of this study was to analyze mutations in the BLV LTR region and tax gene to determine their association with transcriptional activity. LTRs were obtained from one hundred and six BLV isolates and analyzed for their genetic variability. Fifteen variants were selected and characterized based on mutations in LTR regulatory elements, and further used for in vitro transcription assays. Reporter vectors containing the luciferase gene under the control of each variant BLV promoter sequence, in addition to variant Tax expression vectors, were constructed. Both types of plasmids were used for cotransfection of HeLa cells and the level of luciferase activity was measured as a proxy of transcriptional activity. Marked differences in LTR promoter activity and Tax transactivation activity were observed amongst BLV variants. These results demonstrate that mutations in both the BLV LTR and tax gene can affect the promoter activity, which may have important consequences on proviral load, viral fitness, and transmissibility in BLV-infected cattle.

MicroRNA-361-3p is a potent therapeutic target for oral squamous cell carcinoma.

MicroRNAs (miRNAs) can act not only as tumor suppressor genes but also as oncogenes. Oncogenic miRNAs (oncomiRs) could therefore provide opportunities for the treatment of human malignancies. Here, we aimed to identify oncomiRs present in oral squamous cell carcinoma (OSCC) and addressed whether targeting these miRNAs might be useful in treatment for cancer. Functional screening for oncomiRs in a human OSCC cell line (GFP‐SAS) was carried out using the miRCURY LNA microRNA Knockdown Library – Human version 12.0. We identified a locked nucleic acid (LNA)/DNA antisense oligonucleotide against miR‐361‐3p (LNA‐miR‐361‐3p) which showed the largest degree of growth inhibition of GFP‐SAS cells. Transfection with a synthetic mimic of mature miR‐361‐3p resulted in an approximately 20% increase in the growth of GFP‐SAS cells. We identified odd‐skipped related 2 (OSR2) as a miR‐361‐3p target gene. Transfection of GFP‐SAS cells with LNA‐miR‐361‐3p caused a significant increase in the expression levels of OSR2. Cotransfection of a OSR2 3′‐UTR luciferase reporter plasmid and LNA‐miR‐361‐3p into GFP‐SAS cells produced higher levels of luciferase activity than in cells cotransfected with the LNA‐nontarget. We assessed the effect of LNA‐miR‐361‐3p on the in vivo growth of GFP‐SAS cells. We found that LNA‐miR‐361‐3p significantly reduced the size of s.c. xenografted GFP‐SAS tumors, compared to the control group treated with LNA‐NT. Finally, we observed that miR‐361‐3p is overexpressed in OSCC tissues. These results suggest that miR‐361‐3p supports the growth of human OSCC cells both in vitro and in vivo and that targeting miR‐361‐3p could be a useful therapeutic approach for patients with OSCC.

Identification of inhibitors of dengue viral replication using replicon cells expressing secretory luciferase

Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z′ factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.

Ultrasound and Microbubble Gene Delivery for Targeting Altered Placental MicroRNAs in Preeclampsia

Ultrasound (US) and microbubble (MB) gene delivery has attracted growing interest as a clinically applicable gene therapy (GT). Though preclinical studies have investigated the system in various tissues, there is limited research in targeting the placenta. This is a potential therapeutic strategy for preeclampsia (PE), which has an underlying genetic basis and ineffective management strategies. Differentially expressed placental microRNAs (miRNAs) in PE may represent suitable targets for GT. Microbubbles (SonoVue) and plasmid (pGL3 or pGL4.13) were administered systemically to CD1 mice, followed by exposure of the heart to US (H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Luciferase assays were performed to evaluate gene transfection. Significantly differentially expressed placental miRNAs in PE patients were identified as candidates based on detection by three or more screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas. In trial 1, low levels of luciferase activity were detected in the heart of treatment mouse 1, 2 and 3. In trial 2, luciferase activity was evident in the atria of treatment mouse 2. In trial 3, higher luciferase activity was detected in the ventricles of the treatment mouse and activity was also detected in the atria. The literature review identified eight candidate miRNAs. MiR-223 (1.46-fold increase) and miR-181a (0.81-fold decrease) were significantly differentially expressed in PE rat model placentas. MiR-223 and -181a may represent targets for US and MB gene delivery. Future studies will apply the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model.

Anti-inflammatory functions of the CDC25 phosphatase inhibitor BN82002 via targeting AKT2

This study presents BN82002 as an anti-inflammatory drug candidate. It was found that BN82002 inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 cells and peritoneal macrophages that were activated by toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). BN82002 dose-dependently down-regulated mRNA levels of nitric oxide synthase, tumor necrosis factor-α, and cyclooxygenase-2. The nuclear translocation of nuclear factor (NF)-κB (p65 and p50) was also blocked by BN82002 in RAW265.7 cells stimulated by LPS. According to reporter gene assay performed with NF-κB construct, BN82002 clearly reduced increased level of luciferase activity mediated by transcription factor NF-κB in LPS-treated RAW264.7 cells and in MyD88- and AKT2-overexpressing HEK293 cells. However, BN82002 did not inhibit NF-κB activity in AKT1- or IKKβ-overexpressing HEK293 cells. NF-κB upstream signaling events specifically targeted AKT2 but had no effect on AKT1. The specific target of BN82002 was Tyr-178 in AKT2. BN82002 bound to Tyr-178 and interrupted the kinase activity of AKT2, according to a cellular thermal shift assay analysis of the interaction of BN82002 with AKT2 and an AKT2 mutant (Tyr-178 mutated to Ala; AKT2 Y178A). These results suggest that BN82002 could reduce inflammatory pathway by controlling NF-κB pathway and specifically targeting AKT2.

Genetic variations in miR-125 family and the survival of non-small cell lung cancer in Chinese population.

To investigate the associations between the functional single nucleotide polymorphisms (SNPs) in the miR‐125 family and the survival of non‐small cell lung cancer (NSCLC) patients, we systematically selected six functional SNPs located in three pre‐miRNAs (miR‐125a, miR‐125b‐1, miR‐125b‐2). Cox proportional hazard regression analyses were conducted to estimate the crude and adjusted hazard ratios (HRs) and their 95% confidence intervals (CIs). Reporter gene luciferase assay was performed to examine the relationship between the SNPs and transcriptive activity of the miRNAs. The expression of miRNAs in different cells was detected using quantitative real‐time PCR assay. We found that rs2241490 (upstream of miR‐125b‐1, G > A, adjusted HR = 1.24, 95%CI = 1.05‐1.48, P = 0.014, in dominant model; adjusted HR = 1.18, 95%CI = 1.03‐1.35, P = 0.014, in additive model), rs512932 (upstream of miR‐125b‐1, A > G, dominant model: adjusted HR = 1.25, 95%CI = 1.05‐1.48, P = 0.013) and rs8111742 (upstream of miR‐125a, G > A, dominant model: adjusted HR = 0.84, 95%CI = 0.71‐1.00, P = 0.047) were associated with the prognosis of 1001 Chinese NSCLC patients. The combined analysis of the three SNPs related the number of risk alleles (rs2241490‐A, rs512932‐G and rs8111742‐G) to death risk of NSCLC in a locus‐dosage mode (P for trend <0.001). Furthermore, luciferase reporter gene assay showed significantly higher levels of luciferase activity with rs512932 variant G than that with A allele in 293T, SPC‐A1 and A549 cell lines. Besides, miR‐125b was highly expressed in lung cancer cells than the normal lung cell. Our study indicated that genetic variations in miR‐125 family were implicated in the survival of NSCLC patients. Larger population‐based and functional studies are needed to verify these findings.

Association between MEG3/miR-181b polymorphisms and risk of ischemic stroke

BackgroundRecent evidence suggests that long non-coding RNAs (lncRNAs) are key regulators in the pathological process of ischemic stroke (IS). Maternally expressed gene 3 (MEG3) was observed to be up-regulated in IS, acting as a competing endogenous RNA for miR-181b to regulate ischemic brain injury. The purpose of this study was to evaluate the association of tagSNPs in MEG3 (i.e., rs7158663 and rs4081134) and miR-181b rs322931 with IS risk.MethodsGenomic DNA was extracted from blood samples of 509 patients with IS and 668 healthy controls. Genotyping of MEG3 rs7158663, rs4081134, and miR-181b rs322931 was performed by TaqMan assay. The transcriptional activity was measured using the Dual-Luciferase Reporter Assay kit.ResultsSingle-site analysis revealed a significantly higher risk of IS being associated with miR-181b rs322931 CT and CT/TT genotypes (CT vs. CC: adjusted OR = 1.48, 95% CI: 1.13–1.95, P = 0.005; CT/TT vs. CC: adjusted OR = 1.52, 95% CI: 1.17–1.97, P = 0.002). Combined analyses revealed that combined genotypes (rs7158663 GG + rs322931 CT/TT and rs7158663 AG/AA + rs322931 CT/TT) increased IS risk compared to genotypes of rs7158663 GG + rs322931 CC. Stratification analyses showed that patients carrying miR-181b rs322931 CT/TT genotypes had higher levels of low-density lipoprotein cholesterol (LDL_C) (P = 0.01). Moreover, results from logistic regression analysis showed that rs322931 CT/TT genotypes were risk factors besides hypertension, total cholesterol, triglyceride, and LDL_C. Further dual-luciferase reporter assay showed that the rs322931 T allele had lower levels of luciferase activity than the rs322931 C allele.ConclusionThese findings indicate that miR-181b rs322931 may singly or jointly contribute to the risk of IS.

A Calsequestrin Cis-Regulatory Motif Coupled to a Cardiac Troponin T Promoter Improves Cardiac Adeno-Associated Virus Serotype 9 Transduction Specificity.

Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.

PG201 protects mice from experimental autoimmune encephalomyelitis via suppression of effector T cell activation

BackgroundPG201 is a botanical formulation, approved as an ethical drug (ETC) phytomedicine for treatment of patients with osteoarthritis in Korea, following satisfactory phase II and phase III studies. This phytomedicine was previously been shown to possess significant anti-inflammatory activities, presumably via the control of Th1 and Th17 cells in animal models and in vitro cell culture systems. PurposeIn this study, the possibility of using PG201 to treat multiple sclerosis was explored. MethodsIn vitro, the effect of PG201 on the differentiation of CD4+ T cells was investigated. To test the effects of PG201 in vivo, a mouse experimental autoimmune encephalomyelitis (EAE) model was used. ResultsIt was found that PG201 treatment decreased the frequency of both CD4+T-bet+ and CD4+RORγt+T cells. In addition, the production of interferon- gamma (IFN-γ) and interleukin-17 (IL-17) from respective Th cells was highly reduced. The data from western blots showed that the amount of phosphorylated c-Jun, but not that of p65, was decreased by PG201. Consistently, the level of luciferase activity was downregulated by PG201 in activator protein 1 (AP-1) reporter plasmid assays. In mice pretreated with PG201, the day of onset was delayed and clinical symptoms of EAE were significantly improved in a dose-dependent manner. Consistent with these results, the number of infiltrated cells and the expression level of pro-inflammatory molecules were decreased. ConclusionThese findings indicate that PG201 may exert strong immunomodulatory effects in the EAE model via suppression of T cell activation, and that PG201 is a therapeutic reagent for the treatment of multiple sclerosis.

Functional rs4693608 SNP is Located into Strong Hpse Gene Intron Enhancer and Thereby Influences to the Ability of Nuclear Proteins Binding and Activity of This Enhancer in Broad Number of Hematological Cell Lines and ALL/AML Primary Samples

Introduction: Heparanase (HPSE) is an endo-glucuronidase degrading heparan sulfate and playing important roles in angiogenesis, tumor metastasis, inflammation and autoimmunity. The enzyme also regulates histone methylation by binding to target gene regulatory regions. Our previous studies indicated that HPSE gene SNP rs4693608 significantly correlated with the expression level of heparanase among healthy persons. Furthermore, the rs4693608 SNP showed significant association with i) the risk of acute and extensive chronic GVHD, ii) HPSE gene expression both before and after chemotherapy administered as pre-hematopoietic stem cell transplantation conditioning, iii) the time of neutrophils and platelets recovery and iv) HPSE gene expression after lipopolysaccharide (LPS) treatment. Other studies revealed correlations between rs4693608 and sinusoid obstruction syndrome, and with bone morbidity and outcome of multiple myeloma.Methods: In the present study we cloned 440bp fragment of the HPSE gene (intron 2) that includes rs4693608 SNP using Pgl4.26 minimal promoter vector. Reporter activity of luciferase was detected in U937, KG1, K562, RPMI8226, and CAG cell lines. In addition, nuclear protein extracts from a broad range of malignant cell lines as well as from primary samples of patients with acute myeloid (AML) and lymphatic (ALL) leukemia and normal healthy individuals were subjected to binding to 30bp oligonucleotides probe representing the enhancer region of either the A allele or the G allele of rs4693608 SNP by EMSA (electrophoretic mobility shift assay).Results:Our results indicate that the 440bp fragment of the HPSE gene exhibits enhancer activity in both the sense and antisense directions of the HPSE gene. Relative luciferase activity was higher in constructs with antisense direction of the enhancer. Moreover, constructs that included the allele G revealed elevated level of luciferase activity in comparison to constructs that included the A allele. Analysis of healthy control samples using EMSA identified gel shift bands for both allelic probes. However, the affinity of the complex to the G allele probe was significantly higher in comparison to the A allele probe (Fig.1A). The DNA-protein complexes were shifted significantly more in malignant cell lines and primary leukemic samples as compared to normal control samples (Fig.1A). Moreover, there were additional nuclear-protein complexes that bind to the A allele probe (Fig.1B). Notably, at least 5 different DNA-protein complexes connected to allele A were found. In order to identify them we used bioinformatics analysis to predict proteins in the DNA/protein complex, followed by super-shift assay. At least three proteins were identified: β-glucocorticoid receptor, Yin Yang1 (YY1) transcription factor, and CCAAT/enhancer-binding protein-α (C/EBPα), which are part of this structured nuclear protein-binding complex (Fig.1B-C).Conclusions:The study demonstrated that HPSE rs4693608 SNP is located in a strong enhancer. A to G alteration leads to a different enhancer activity as a result of unique DNA-protein complex which binds to DNA regulatory elements in the enhancer. The composition of the DNA-protein complex which binds to the G allele, shifted similarly in all cell lines and primary leukemic samples but not in normal samples. At least 5 different variations of DNA-protein complexes connected to allele A were found, including β-glucocorticoid receptor, YY1 and C/EBPα. Precise identification of the specific proteins that bind to the unique enhancer region will allow modification of the enhancer activity that accelerates the expression of HPSE and thereby influences downstream processes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-146a polymorphism correlates with lung cancer risk in Chinese nonsmoking females.

This study provides evidence that the common rs2910164 polymorphism in miR-146a strongly correlates with lung cancer risk in nonsmoking females in northeast China. The genotypes of miR-146a rs2910164 were determined in 1131 patients with lung cancer and 1003 healthy control subjects. Tissue samples were used to evaluate the association between miRNA expression and lung cancer risk as well as the correlation between rs2910164 genotypes and miR-146a expression. The secondary structures of the wild-type and variant miR-146a sequences were predicted, and luciferase-based target assays were used to test whether miR-146a bound to tumor necrosis factor receptor associated factor 6 (TRAF6) mRNA. Individuals carrying heterozygous CG genotype of miR-146a rs2910164 had less risk of lung cancer than those carrying homozygous wild CC genotype (OR = 0.76, 95% CI = 0.60-0.98, P = 0.032). We found no significant association between miR-146a expression and lung cancer risk. MiR-146a expression differed in those carrying the CC genotype as compared with the CG or the GG genotype (P = 0.032 and 0.001), and the secondary structure of the C allele differed slightly from the G allele. Significantly lower levels of luciferase activity were observed when the TRAF6 3′UTR was cotransfected with miR-146a-3p carrying the rs2910164 C allele (P = 0.001). Thus, miR-146a rs2910164 polymorphism may influence susceptibility to lung cancer in Chinese nonsmoking females through targeting TRAF6.